First molecular detection and genetic characterization of porcine circovirus 4 in the Gansu Province of China

Since its initial discovery in the Hunan province of China, genomic DNA of porcine circovirus 4 (PCV4) has been detected in pigs across multiple provinces in China, as well as in South Korea. However, the prevalence of porcine circovirus type 4 in Gansu Province, China, remains unknown. To address this gap, we undertook an extensive study where we gathered 121 clinical samples displaying diverse clinical manifestations from pig farms in Gansu Province between 2022 and 2023. Employing a real-time fluorescence quantification method, we identified the presence of PCV4 genome. Out of the 121 clinical samples analyzed, 13 samples tested positive for PCV4, resulting in a positive rate of 10.74% (13/121). This finding confirms the presence of PCV4 in pig farms within Gansu Province, China. Furthermore, we successfully sequenced and analyzed the complete genomes of two distinct PCV4 strains, comparing them with 60 reference sequences archived in the GenBank database. The results revealed a high nucleotide homology (98.2–98.8%) between the strains obtained in this study and the PCV4 reference strains, indicating a relatively low evolutionary rate of the PCV4 genome. Phylogenetic analysis revealed that two strains in this study belong to PCV4a and PCV4c. As far as we know, this study marks the inaugural report on the molecular identification and genomic attributes of PCV4 in Gansu Province, China, offering valuable insights for devising preventive and control strategies against this emerging virus.

The aim of this study was to assess the occurrence of PCV4 in pig farms situated in Gansu, China, during the period from 2022 to 2023.Furthermore, complete genomes were amplified to explore the genetic variability of the virus present in Gansu Province.

Samples collection and viral genome extraction
A total of 121 samples were collected from 14 pig farms in 8 cities (Jinchang, Wuwei, Baiyin, Lanzhou, Dingxi, Gannan, Tianshui and Pingliang) during 2022-2023 (Table 1).Among the collected samples, 89.26% (108/121) were obtained from diseased pigs exhibiting various clinical signs, including respiratory, enteric, and PDNS, for diagnostic purposes.The remaining 10.74% (13/121) were collected from healthy animals for infectious disease surveillance.The diseased pigs were humanly euthanized with an overdose of pentobarbitalum natricum [Natriumpentobarbital 20% 1 40 mg/kg iv in the V. jugularis externa].Then, samples were collected.In healthy pigs, venous blood and swab paper were collected after intramuscular injection of ketamine 15 mg/kg.The sample types encompassed brain, kidney, spleen, lung, serum, oral fluid, feces, lymph node, tonsil, nasal swab, placenta, intestine, and semen.All experimental procedures were reviewed and approved by the Henan University of Urban Construction Animal Care and Use Committee (license number HNXK (Henan) 2015-0001).All samples were collected under the supervision of veterinarians from College of Life Science and Engineering.All efforts were made to minimize their suffering throughout the experiment.
Each sample was individually processed by mixing it with phosphate-buffered saline (PBS) in a sterile 1.5-mL microcentrifuge tube.Subsequently, it underwent three cycles of freezing and thawing before being centrifuged at 12,000 ×g.The resulting supernatant was either utilized for immediate viral genome extraction or stored at -80˚C until further use.DNA and RNA extraction procedures were carried out using the FastPure Viral DNA/RNA Mini Kit from Vazyme Biotech Co., Ltd., Nanjing, China, in accordance with the manufacturer's guidelines.The viral genome was then identified through a real-time PCR assay based on SYBR Green І, as detailed in a previous publication [19].Meanwhile, other common viruses, including porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), Porcine deltacoronavirus (PDCoV), swine acute diarrhea syndrome-coronavirus (SADS-CoV) pseudorabies virus (PRV), porcine circovirus 2 (PCV2) and porcine circovirus 3 (PCV3) were also detected using PCR or RT-PCR assay as previously described [24][25][26][27][28][29].

Complete genome sequencing
As described previously [30], the complete genome of PCV4 was sequenced employing three sets of overlapping primers (S1 Table ).The PCR mixture was composed of 10 μL of KOD OneTM PCR Master Mix (Toyobo (Shanghai) Biotechnology Co., Ltd., China), 0.5 μL each of forward and reverse primers (at 10 mM concentration), 1 μL of template DNA, and 8 μL of double-distilled water.The amplification parameters for these three primer pairs were as follows: 98˚C for 30 s; 30 cycles of 98 ˚C for 10 s, 60˚C for 5 s, and 68˚C for 5 s.According to the manufacturer's instructions, the PCR amplification products were purified using MagExtrac-tor™ -PCR & Gel Clean up-(Toyobo, Shanghai, China) and cloned into the pMD18-T vector (Takara, Dalian, China) for constructing recombinant plasmid.The recombinant plasmids propagated in Escherichia coli DH-5α cells (Tolo Biotechnology, Shanghai, China).The confirmed positive clones were dispatched to Sangon Biotech Co., Ltd (Shanghai, China), for sequencing.The complete genome was then assembled utilizing the EditSeq and Megalign programs within the LaserGene software package (DNASTAR, Inc., Madison, WI).

Sequence alignment and phylogenetic analysis
The complete genomes of PCV4 strains in this investigation underwent analysis alongside 60 reference strains cataloged in GenBank.Information regarding the reference strains was summarized in S2 Table .Nucleotide and deduced amino acid sequences were aligned using the Clustal W method in the MegAlign program.Molecular Evolutionary Genetics Analysis (MEGA) software (version 7.0) was applied to construct phylogenetic tree by the neighbourjoining method with a p-distance model, and a bootstrap of 1000 replicates.

Results and discussion
Previous studies have shown that porcine circovirus is prevalent in swine production worldwide.PCVAD caused by PCVs, especially PCV2 and PCV3, has caused significant economic losses to the global pig industry.Therefore, PCV4 has attracted a lot of attention as an emerging porcine circovirus in 2019.Moreover, a recent study suggests that PCV4 is pathogenic to piglets [23].However, information on the prevalence of PCV4 in Gansu Province, China is lacking.Therefore, to fill this gap, this study conducted an epidemiologic survey of PCV4 in Gansu Province.
Currently, there are various molecular epidemiology studies on PCV4 in pigs across certain provinces of China and Korea, reporting positivity rates ranging from 1.6% to 45.39% [31,32].In this study, 121 samples were collected from 121 pigs with different clinical manifestations in Gansu Province, China.Of these, PCV4 was identified in 10.74% (13/121) of samples, which was far lower than that of PCV4 45.39% (69/152) in Henan Province reported by Hou et al but higher than that of PCV4 1.6% (27/1683) in Inner Mongolia Province [31,32].Details of the 13 positive samples were summarized in Table 2. Of the 13 positive samples, 38.46% (5/13) originated from kidneys, 23.08% (3/13) from intestines, 23.08% (3/13) from lungs, and 15.38% (2/13) from oral fluids.Other studies have demonstrated the presence of PCV4 DNA in nearly all tissues, including sera, heart, liver, spleen, lung, kidney, lymph nodes, tonsils, intestines, and brain, in both diseased piglets and healthy animals [31][32][33].These results indicate that PCV4 has a broad tissue propensity to facilitate horizontal and vertical transmission.The clinical samples were collected from 8 cities during 2022-2023.PCV4 was identified from samples of 3 cities (Lanzhou, Wuwei, Baiyin) (Table 1).PCV4 was not detected in other cities, possibly due to the limited number of samples available.The 13 positive samples came from 13 diseased pigs at different stages of growth, presenting symptoms such as respiratory disease, diarrhea, and PDNS.These findings suggested that PCV4 DNA could be detected in Gansu Province with a low positive rate and limited geographical distribution.In my opinion, prompt measures should be implemented to mitigate the potential threat posed by PCV4 to the pig industry in Gansu Province.
Besides the infection of PCV4 described above, we investigated the coinfection of PCV4 with other pathogens, such as PCV2, TGEV, SADS-CoV, PCV3, PRV, PDCoV, PEDV and PRRSV.In brief, 23.08% (3/13) of PCV4-positive samples were coinfected with PCV2, 23.08% (3/13) were coinfected with PCV3, 15.38% (2/13) were coinfected with PRRSV and 23.08% (3/ 13) were coinfected with PEDV.Interestingly, one sample was positive for PCV2, PCV3 and PCV4.The remaining 3 samples were PCV4 positive with no other pathogens detected, and two of which did not show clinically obvious signs.These samples were submitted for diagnosis.Unfortunately, due to the fact that the tissue samples were not fixed with 4% paraformaldehyde, we were unable to perform hematoxylin and eosin (H&E) staining and immunohistochemical assays.Consequently, we could not investigate the potential association between PCV4 and pathological changes.Currently, we are making efforts to isolate PCV4 strains.If successful, animal return experiments will compensate for these regrets.
To gain insights into the genetic characteristics of PCV4 in Gansu Province, we successfully obtained and deposited two distinct complete genomes (GS2022-BY and GS2022-LZ) in the GenBank database.These genomes have been assigned the accession numbers OQ970016 and OQ970017, respectively.The reasons why only two strains have been sequenced were as follows: First, the viral load of some samples was low.Second, the genome of PCV4 has a stemloop structure that makes it difficult to amplify.Compared to the reference strains, no base insertions and deletions occurred in the whole genomes of GS2022-BY and GS2022-LZ with a size of 1770 nt.Then, GS2022-BY and GS2022-LZ were analyzed with 60 reference strains deposited in GenBank (S2 Table ).Sequence analysis indicated that the GS2022-BY and GS2022-LZ shared 98.6% whole genome nucleotide identity, and these two strains showed high identities of 97.9-99.5% to the genomes of the reference strains.These reference strains were detected in different species (pigs, dogs, foxes and cows) and in different geographical distributions (several provinces in China and Korea) [30,[34][35][36].The genomes of these reference strains date back to as early as 2012 [32].The high homology observed between the strains in this study and reference strains from various sources suggests that the PCV4 genome is highly conserved.
The limited number of existing reference strains and the high genomic identity between these strains pose challenges in classification of PCV4.Nevertheless, based on several suggested classification schemes [19,30], PCV4 has been categorized into either two (PCV4a and PCV4b) or three primary clades (PCV4a, PCV4b, and PCV4c).As previously described, we constructed a NJ phylogenetic tree (Fig 1) [30], keeping all parameters the same except for an expanded set of reference strains.According to the proposed clade classification and amino acid marker positions outlined by Xu et al. [30], three different genotypes, namely PCV4a, PCV4b and PCV4c were observed in Fig 1 , and the two strains in this study clustered in the proposed PCV4a and PCV4c, respectively.Curiously, the phylogenetic tree suggested that a PCV4 reference strain belonged to PCV4c in Xu et al, whereas in this study it was included in an undefined cluster between PCV4b and PCV4c.Meanwhile, it is difficult to elucidate any potential clade distribution by geographic location, as PCV4 sequences found on different farms in several provinces in China and Korea were represented in more than one clade, while sequences coming from another part of the provinces (e.g.Inner Mongolia) were found in a single group.These observations suggested that the genotype classification proposed by Xu et al. (2022) might have limitations.It is recommended to prioritize genetic distance and phylogenetic clustering as the primary criteria [37].Additionally, other factors such as the number of sequences within clusters, host and geographic clustering, concordance between different genomic regions, and analysis methods should be considered to develop an effective classification system for research and diagnosis [37].Therefore, the genotype classification scheme of PCV4 warrants further study.
There were no amino acid insertions and deletions in the Rep and Cap proteins of these 62 strains.All identified amino acid mutations in Rep and Cap of the 62 PCV4 strains were  [30].However, two PCV4 reference strains (KU-02010 and KU-02011) from Korea, as well as one reference strain (PCV4-PY) from Henan Province, China, were classified as PCV4a in the phylogenetic tree, yet their Cap protein's 212th position contained M and P, respectively, instead of L. This indicated that the marker positions suggested by Xu et al. might not be universally applicable.GS2022-BY and GS2022-LZ also contained the essential elements for the replication of circoviruses in pig-origin PCV4 strains predicted by Nguyen et al [34], such as the origin of DNA replication, endonuclease and helicase.
The arginine-rich region known as the nuclear localization signal (NLS) within the circovirus genus plays a role in facilitating the nuclear localization of the viral genome [38][39][40].Recently, it was reported that the NLS of PCV4 Cap is located at the N-terminal residues 1-20 [41], which was also observed in GS2022-BY and GS2022-LZ.In the PCV4 Cap, five potential linear B cell epitopes with high antigenicity were predicted by Wang et al [42], including epi-

Conclusion
In conclusion, to the best of our knowledge, this study is the first to report the detection of PCV4 DNA in swine farms in Gansu Province with a low positivity rate.Furthermore, we successfully obtained the complete genomes of two PCV4 strains, which were classified into PCV4a and PCV4c clusters.These results enhance our comprehension of the prevalence and genomic characteristics of PCV4 in pig farms located in Gansu Province, China.

Fig 1 .
Fig 1.The neighbor-joining tree was constructed based on the complete genome of 62 PCV4 strains with a p-distance model and bootstrapping at 1,000 replicates.Different colors represent different genotypes.Blue, fuchsia and purple represent the three genotypes of PCV4 (PCV4a, PCV4b and PCV4c) proposed by Xu et al (2022), respectively, while red represents undefined genotype.https://doi.org/10.1371/journal.pone.0293135.g001 tope A: 72-88, 104-112, epitope B: 122-177, epitope C:199-205, and epitope D:219-225 (Fig 2).Out of the 50 amino acid mutations found in the Cap protein of the 62 PCV4 strains, 16 were situated within the anticipated epitope region.These mutations might potentially result in a modified antigenic profile of the Cap protein, warranting additional investigation.

Fig 2 .
Fig 2. All amino acid mutations in Rep and Cap.Different background colors indicate different genotypes proposed by Xu et al (2022), respectively.The red open box showed the amino acid markers of genotypes proposed by Xu et al (2022).The amino acid positions in the gray region were included in the potential linear B-cell epitopes predicted by Wang et al (2021).The PCV4 strain in this study was marked by solid circles.https://doi.org/10.1371/journal.pone.0293135.g002

Table 2 . Origin, clinical manifestation and detection results of 13 PCV4 positive clinical samples from the Gansu Province of China during 2022-2023. Sample name Collection date Sample type Geographical location Farm Growth stages Clinical symptoms PEDV TGEV PDCoV SADS-CoV PRV PCV2 PCV3 PRRSV
Note:The complete genomes of GS2022-BY and GS2022-LZ were acquired from sample 1 and 7 marked with *.No represents no obvious clinical manifestations.https://doi.org/10.1371/journal.pone.0293135.t002